Using Clinical Biologic Correlates To Inform HSV Trial Design Through this Program Project grant we successfully generated and characterized several new, promising genetically engineered herpes simplex viruses (HSV) that are candidates for evaluation in clinical trials of patients with malignant glioma. These viruses were extensively studied and characterized in both in vitro and in vivo models to demonstrate proof-of-principle of safety and efficacy. Increased efficacy was provided by (i) expression of certain foreign genes and (ii) adjunctive therapy with radiation. This project will exploit our clinical trial experience in Phase I studies of G207, a first generation engineered oncolytic herpes simplex virus (oHSV) in the treatment of gliomas. We have developed extensive experience in the first use of a genetically engineered HSV for the treatment of human glioma in three Phase I clinical trials, with an upcoming study of a human IL-12 expressing engineered HSV-1 (M032) to begin imminently. We have collected a variety of tissue specimens to delineate the biologic response of human glioma patients treated with G207. We propose to use these human specimens for the following: AIM 1 will determine if IL-12 expression by oncolytic HSV produces differences in the glioma tumor microenvironment and viral replication that impact tumor response. Evaluation of data from our G207 trials and upcoming M032 trial will be undertaken; response to therapy and corresponding findings will be evaluated for potential correlations. AIM 2 will determine whether HSV-treated tumors have SR, NSR or nu87 gene expression profiles defined by Project 2 and whether any of these GEPs are predictive of tumor response to treatment with ILI2 expressing OHSV, as well as identifying additional GEPs that are associated with poor outcome after treatment with IL- 12-expressing oHSV. Preclinical models emulating these responses will be developed to confirm the predictability of response to different viral constructs and adjunctive therapies. AIM 3 will determine the impact of glioma progenitor cells (GPC) on response to oncolytic HSV therapy by contrasting the fraction of GPC present with the response to virus therapy. Similarly we will assess the effects of IL-12 expression on GPC response. Aim 4 will serve as a planning aim for the next oncolytic HSV (after M032) to enter clinical trials that emerges from this Program Project. The aim includes decision analysis selection of the virus to be used, along with any adjuvant therapies; initiation of pretrial animal studies; and development of a clinical protocol, production of cGMP virus, as well as application for additional funding to actually perform the trial. RELEVANCE (See instructions): We have undertaken 3 Phase I clinical trials with a first generation oncolytic HSV in patients with malignant glioma and our results have confirmed our observations that tumor gene expression profile (GEP) defines susceptibility or resistance. Studies proposed here will analyze tumor tissues from these patients and from those to be treated with our second generation oHSV, expressing ILI 2. From these biologic studies, correlation of tumor GEP with response to oHSV will inform design of new oHSV that can overcome glioma resistance factors and broaden applicability of oHSV for efficacious anti-glioma therapy.